In practice, peptides have been synthesized in quantities ranging from milligrams to less than 10 grams. Existing machines have proven more reliable in the 0.1-1 gram scale. Syntheses can be carried out by stepwise addition of amino acids or by fragment coupling reactions under inert gas or dry atmospheric conditions. Reagents can be transferred by inert gas pressure, by pump or by vacuum. Reagent delivery in the two currently marketed SPPS instruments (namely, Model 990 of Beckman located in California and Model 250 of Vega Biochemical located at Tucson, Ariz.) is accomplished by transferring reagent from a reservoir to a premetering flask(s). The volume of reagent is measured by monitoring with a photocell. From the premetering flask(s) the reagent is then delivered to the reaction vessel by activating a valve. The disadvantage to this system is that the premetering flask must have input conduits from many reagent reservoirs. This common connection can allow contamination of not only the solvent being delivered, but also of the reagent reservoirs themselves by vapors or liquid from other reagent reservoirs. Currently available solid phase peptide synthesizer (hereafter SPPS) machines known to me have up to 12 amino acid reservoirs and up to 11 solvent and other reagent reservoirs are capable of synthesizing only one peptide at a time.
Currently available SPPS machines of which I am aware thus have certain disadvantages which the method and apparatus of the present invention is intended to overcome.
Accordingly, the present invention contemplates an apparatus and method not only capable of performing the functions of currently marketed SPPS models, but which also possess several advantageous improvements as set forth below.
The inventive apparatus and method provide for synthesis of more than one peptide to be carried out under the same condition(s) simultaneously, but with only one set of reagents being required. Two methods are contemplated. In a single path method a multi-chambered vessel or multicolumns are used. In a multi-path method each synthesis is carried out in an individual vessel or column. Simultaneous synthesis of more than one peptide at a time has the following advangages.
(a) Cost saving (labor, initial investment in the number of apparatus, space and other related types of expense).
(b) Time saving.
(c) Assurance that if two similar peptides are synthesized simultaneously the analogs will be essentially identical, except for the intended differences.
The inventive method and apparatus also facilitate synthesis of peptides of higher quality with more rapid coupling and synthesis than those synthesized on prior machines known to me because of the following reasons:
(A) Less chance of cross contamination because of the following:
(a) The manner in which reagents are transferred to the reaction vessel.
(b) Flushing of the lines after each use.
(c) Minimizing the amount of acid or base left behind in the reaction vessel, which reduces the side products and/or racemization.
(d) Preactivation of protected amino acids before addition to peptides on the resin, which reduces contact time and possible side reaction of the peptide with reagents and other additives.
(e) Addition of preactivated amino acids to increase the rate of coupling reaction and decrease the side products.
(B) Rapid transfer of reagents to the reaction vessel(s) or draining from the reaction vessel(s), which is especially advantageous for large scale production because of uniform treatment of the resin in the reaction vessel.
(C) Adaptability to control of temperature and pH, which should also help purity, increase rates of reaction, and otherwise improve the synthesis.
The inventive apparatus and method also are particularly flexible in enabling easily and precisely controllable synthesis of a wide variety of peptides and are adapted for control manually, by hard wired circuitry and by a microprocessor having a master program containing from one to over 200 files. Individual synthetic procedures can be stored in each file. The output from the controller can be connected to a printer for a permanent record of the synthesis in progress. In each file any reagent in any order one wishes can be chosen. This flexibility provides the following advantages.
(a) Number of reagents is not limited.
(b) Order of reagents is not restricted.
(c) Type of solid supports is not limited.
(d) Number of amino acids is not limited.
(e) No limitation on new types of protecting groups, or coupling methods.
(f) Never need to write a program or load a program.
(g) A permanent record of the synthesis is produced.
The objects and purposes of the invention are met by providing a method and apparatus for peptide synthesis in which a plurality of supply valves are arranged in series to define a line for supplying successive reagents to a reaction vessel in which a peptide is to be synthesized. A corresponding plurality of containers contain respective reagents and each, by actuation of its corresponding valve, is connectable through downstream ones of the series of valves to supply its respective reagent to the reaction vessel. For cleaning or reacting with peptide starting material therein further valving permits draining of the reaction vessel to waste and, between applications of successive reagents to the reaction vessel, cleaning of the line of valves by flushing with a cleaning reagent sent from the upstream end of the series of valves through the line to waste. In modified embodiments, the plurality of reagent containers and series line of valves supplies reagents to multiple reaction vessel chambers in such a way that the multiple chambers may simultaneously be occupied by the same reagent in one peptide synthesis step and by several differing reagents, delivered thereto in sequence, in a differing peptide synthesis step, to enable simultaneous production of similar but different peptide analogs.
Other objects and purposes of this invention will be apparent to persons acquainted with apparatus of this general type upon reading the following specification and inspecting the accompanying drawings.